Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vasculature with high morbidity and mortality that frequently results in right ventricular (RV) failure and death. Even with the clinical success of drugs, such as prostacyclins and endothelin receptor antagonists, the five-year survival in PAH remains ~ 50%. Therefore, there is an unmet medical need for new approaches to treat/prevent this disease. This puts into context recent results that identified vascular endothelial growth factor receptor 3 (VEGFR3) signaling as protective in PAH. Specifically, it was determined that endothelial specific knockout of VEGFR3 in mice resulted in severe PAH secondary to abnormal pulmonary vascular remodeling. The clinical relevance of these findings was highlighted by a loss of VEGFR3 expression in lung tissue from patients with PAH. These findings suggest that loss of VEGFR3 expression and/or activity is causally associated with the pathobiology of PAH and that interventions that increase the expression of this receptor, or which phenocopy VEGFR3 activity, may have utility in the treatment of PAH. Thus, defining the targetable nodes in VEGFR3-regulated signaling pathway(s) amenable to pharmaceutical intervention is a primary focus of our research. In our preliminary studies arising from my K08 award, we have shown that knockout of -arrestin 1 (arr1), an adapter protein that modulates the signaling of G protein-coupled receptors (GPCRs), results in severe pulmonary hypertension and RV dysfunction in mice. It was also determined that (a) activation of VEGFR3 was dramatically attenuated in arr1 knockout mice and (b) arrs bind to and modulate VEGFR3 activity, findings that are consistent with the hypothesis that arr1 is a mediator of the intracellular actions of VEGFR3. The long-term goal of our research is to identify regulatory nodes in the VEGFR3/arr signaling pathway(s) that can be exploited for the development of new therapeutic approaches to treat PAH. The overall objective of the studies outlined in this application, therefore, is to define the mechanism(s) by which arrs regulate VEGFR3 activity and how downstream signaling events impact pulmonary endothelial function. Our central hypothesis is that arr1 is the primary mediator of VEGFR3 signaling in endothelial cells and that the resultant signaling events prevent the dysregulated pulmonary vascular remodeling that underlies the development of PAH. We plan to test our hypothesis and accomplish the objectives of this application by pursuing the following specific aims: (1) Identify motifs in arrs and VEGFR3 that are critical for their interaction and subsequent signaling. (2) Elucidate arr-mediated signaling pathways downstream of VEGFR3 and the effects of this signaling on endothelial cell function. This project explores a new, innovative signaling pathway that could be exploited to treat PAH, and could also have an impact in other diseases associated with endothelial dysfunction, i.e., heart failure and cancer. Funding of this proposal will also support a future R01 submission focusing on testing the effects of increasing and decreasing arr-mediated VEGFR3 signaling in small animal models of PAH.